Prognostic value of DNA flow cytometry in sympathoadrenal paragangliomas.

OBJECTIVE: To determine whether ploidy patterns are related to prognosis in sympathoadrenal paragangliomas (SAP) using flow cytometry. STUDY DESIGN: DNA flow cytometric analysis of formalin-fixed, paraffin-embedded tumor samples from 36 patients with SAP was performed. Eight cases fulfilled at least one of the following malignancy criteria: (1) extensive invasion of adjacent structures (5 cases), (2) local recurrence (3 cases), or (3) metastases (4 cases). RESULTS: Of the 36 tumors, 22 (61%) showed nondiploid patterns (12 aneuploid, 10 tetraploid). All diploid tumors were benign, while all malignant cases showed nondiploid patterns (P = .0131). The differences between diploid and aneuploid tumors and between diploid and tetraploid tumors, with regard to the malignancy of the disease, were statistically significant (P = .03311 and .01976, respectively). Only one malignant tumor had a DNA index < 1.75 (P = .00259). CONCLUSION: Anomalous DNA ploidy patterns are frequent in SAP, without necessarily implying malignancy. However, diploid DNA content may be a marker of a good prognosis. The likelihood of malignancy is greater in the tetraploid and peritetraploid range.     

应用H.E染色的鼻咽癌细胞涂片进行DNA含量测定和EB病毒基因原位杂交的研究

分析鼻咽癌常规 H.E染色细胞涂片分别作 Feulgen染色检测 DNA含量和原位杂交检测 EB病毒编码 RNAs(EBERs)的效果 ;将常规 HE染色的 9例正常鼻咽细胞涂片和 38例鼻咽癌细胞涂片用 1%酸性酒精褪色 ,然后作 Feulgen染色 ,应用图象分析仪测定涂片细胞 DNA含量 ,并将 38例鼻咽癌细胞病例的阳性涂片进行 EBERs原位杂交检测 ;结果显示正常鼻咽细胞均为 DNA二倍体核型 ,38例癌阳性涂片中呈 DNA二倍体者 6例 ,DNA非二倍体者 32例 (84.2 % ) ,癌细胞核 EBERs阳性者 35例 (92 .1% ) ;结果表明 HE染色的鼻咽癌细胞学涂片经褪色后作 Feulgen染色和原位杂交检测 ,能较满意进行 DNA和 EBERs分析 ,表明常规 H.E染色和褪色处理均不影响 Feulgen染色和 EB病毒原位杂交的质量效果。本检测方法可靠、可行 ,适用于常规染色细胞学样本的回顾性研究和随访研究 ,并可用于鼻咽癌可疑病人的诊断和鉴别诊断     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

Applicability of liquid-based cytology to the assessment of DNA content in cervical lesions using static cytometry

OBJECTIVE: To evaluate the nuclear DNA content of cervical lesions in liquid-based cytologic specimens prepared for static cytometry. STUDY DESIGN: The DNA content of cervical lesions was evaluated in cervical samples prepared with the Autocyte PREP liquid-based cytology system (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.). A series of 47 samples stained with the Papanicolaou method (chronic cervicitis, n = 15; cervical intraepithelial neoplasia [CIN] 1, n = 25; CIN 2, n = 5; CIN 3, n = 2) were collected from consecutive women enrolled in an ongoing screening study at Leonor Mendes de Barros Hospital, S?o Paulo, Brazil, in 2002. Each residual sample was processed according to the Feulgen-thionin method (TriPath Imaging). Ploidy evaluation was performed using the CAS 200 image analysis system and Quantitative DNA Measurement software 3.0 (version 8.1) (Becton Dickinson, San Jose, Califoria, U.S.A.). Cellular ploidy was analyzed from atypical nuclei, and the DNA index was obtained using histograms for interpretation. RESULTS: All chronic cervicitis cases were diploid. Of the CIN 1 cases, 44% were diploid, 12% tetraploid, 32% aneuploid and 12% polyploid (diploid plus tetraploid). CIN 2 lesions were diploid in 60% and aneuploid in 40% of cases, whereas all CIN 3 lesions (100%) were aneuploid. CONCLUSION: The liquid-based cytologic samples proved to be suitable and highly useful for DNA analysis by image cytometry, which was capable of discriminating CIN 3 lesions from CIN 1 and 2 but not CIN 1 from 2 lesions. Aneuploidy was closely associated with CIN 3 lesions.     

Comparison of the intratumor DNA ploidy distribution pattern between differentiated and undifferentiated gastric carcinoma

OBJECTIVE: To compare the differences in intratumor DNA ploidy distribution pattern between differentiated and undifferentiated carcinoma of the stomach. STUDY DESIGN: Two hundred five cases of surgically resected gastric carcinoma were studied, comprising 125 differentiated and 80 undifferentiated carcinomas. DNA ploidy was determined by means of flow cytometry in stepwise sections of the entire tumor. The intratumor DNA ploidy distribution pattern was compared between these two histologic types. RESULTS: Differentiated carcinomas comprised 35 cases of predominantly diploid (28%) and 90 cases of predominantly aneuploid (72%) tumors, while undifferentiated carcinomas comprised 46 cases of predominantly diploid (57.5%) and 34 cases of predominantly aneuploid (42.5%) (P < .01). The frequency of cases showing predominantly aneuploidy among the differentiated carcinomas was significantly higher than among the undifferentiated carcinomas at stages IA and IIIA. The rate of cases showing heterogeneity was lower among the early-stage cases than among the advanced-stage cases in both histologic types. Among the differentiated carcinomas, there were 22 cases that had more than six DNA stemlines in each tumor, whereas among undifferentiated carcinomas, there were six such cases (P < .05). CONCLUSION: There were more cases with high malignant potential among the differentiated carcinoma cases than among the undifferentiated carcinoma cases in the present series.     

Colorectal carcinoma. DNA ploidy pattern and prognosis with reference to tumor DNA heterogeneity.

Nuclear DNA content was measured in 72 colorectal carcinomas using single-cell microspectrophotometry on Feulgen-stained smears. Four samples were analyzed from each tumor. Patients were followed for 41-65 months (average, 53). DNA heterogeneity (both aneuploid and nonaneuploid patterns) was present in 44% of the cases. Sixty-eight percent of the tumors showed an aneuploid DNA pattern in at least one of the samples. Patients with nonaneuploid tumors tended to have a survival advantage over patients with homogeneously aneuploid tumors and demonstrated a significantly longer disease-free survival. The DNA ploidy pattern is of potential value in conjunction with histopathologic prognostic parameters in colorectal carcinoma. Since colorectal tumors exhibit pronounced DNA heterogeneity, multiple samples are required from each tumor to permit a proper evaluation of its DNA pattern. The DNA heterogeneity may represent tumor progression and can partly explain the conflicting results reported concerning DNA pattern and prognosis in colorectal carcinoma.     

DNA ploidy in seminal vesicle cells. A potential diagnostic pitfall in urine cytology

OBJECTIVE: To verify that abnormal DNA ploidy in urine cytology can occasionally be attributed to contamination by seminal vesicle cells. STUDY DESIGN: In the first part of this study, we analyzed the DNA content of six urine cytology specimens containing seminal vesicle cells. In the second part, we evaluated 21 Feulgen-stained prostate core biopsies containing seminal vesicle-type epithelium using a CAS-200 system. DNA index, proliferative activity (S + G2M) and degree of hyperploidy (> 5C) were determined in each case. RESULTS: All six urine cytology specimens were diploid, with all but one containing hyperploid cells (range, 0-16%; mean, 6.3%). Seminal vesicle cells from prostate biopsies showed a broad range of ploidy abnormalities. Ten cases (48%) showed an aneuploid peak, two cases (9%) showed a tetraploid peak, and nine cases (43%) showed only a diploid peak. All but one case showed both an elevation in proliferative activity (mean S + G2M, 24.2%) and some hyperploid cells (mean, > 5C; 4.5%). CONCLUSION: Seminal vesicle cells, although rarely seen in urine cytology, can cause abnormal DNA ploidy measurements. Morphologic criteria remain vital to an accurate cytologic diagnosis.     

Comparison of cytologic and acridine-orange flow-cytometric detection of malignant cells in human body cavity fluids

Flow cytometrically (FCM) derived DNA and RNA profiles were studied in acridine orange (AO)-stained body cavity fluid (BCF) specimens obtained from 78 patients with various solid tissue and hematologic malignancies. The ploidy (DNA index), RNA content (RNA index), proliferative activity (% S + G2M) and DNA and RNA scattergram patterns were tested "double-blind" against the cytologic scoring of specimens as malignant, benign or reactive. It was determined that expression of an "abnormal" RNA index (greater than or equal to 2.8) and an elevated proliferative activity (% S + G2M greater than or equal to 7.4) was dependent on the presence of malignancy; 21 of 22 specimens having those abnormal indices had DNA aneuploidy and were cytologically scored as positive. The AO FCM sensitivity and specificity for detecting malignant cells (when measured against cytology scoring) were 61% and 90%, respectively, using the "abnormal" RNA index and % S + G2M cut-offs together with the cellular DNA aneuploidy marker. By supplementing the cytologic scoring with AO FCM DNA and RNA features, the sensitivity for detecting malignant cells was 94%, as compared to 72% for cytology alone. Two specimens gave false-positive FCM results: a tuberculous effusion with a tetraploid subpopulation and a reactive mesothelial proliferation that was diploid and negative cytologically. Scoring for malignancy based on the visual pattern of the DNA and RNA FCM scattergrams, while showing good correlation for aneuploid specimens, in some cases failed to identify diploid disease. The results demonstrate the usefulness of FCM DNA and RNA analysis for supplementing cytologic examination of BCF specimens for the purpose of detecting malignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of tumor heterogeneity of prostate carcinoma by flow- and image DNA cytometry and histopathological grading.

BACKGROUND: Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. MATERIAL AND METHODS: Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 B?cking's grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow- and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. RESULTS: By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. CONCLUSION: Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.     

Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

Intratumoral variations in DNA distribution patterns in mammary adenocarcinomas

Correlated flow-cytometric (FCM) and microspectrophotometric (MSP) techniques were applied to investigating whether intratumoral variations in the DNA distribution patterns of 21 primary mammary adenocarcinomas can occur. Although neoplastic cell populations with both diploid and tetraploid (i.e., euploid) distribution patterns could be found in varying proportions in some of the tumors, there was no evidence in any tumor nodule for the presence of euploid populations in one part and aneuploid populations in another. This statement was based on the results of the MSP technique, where the assessments were made on cytodiagnostically identified neoplastic cells. Also, when applying the FCM technique the statement was found to be essentially valid; only one of the tumor nodules showed a DNA distribution pattern that, by means of the criteria used in this procedure, was defined as being both euploid and aneuploid. Here, however, the technique consists of assessments made on a great number of microscopically non-identified cells. It was concluded that when conflicting reports are given from different laboratories on the prognostic value of the cytochemically assessed DNA distribution patterns in breast carcinomas, they are not likely to be attributed to intratumoral DNA heterogeneity but, rather, to differences in the methods used and in the criteria applied for the so-called ploidy assessments.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.     

Bone metastases: prognostic applications of cytometric DNA analysis

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.     

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

D. Demirel, N. Akyürek and I. Ramzy
Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions Objective: To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. Methods: The study included 31 cases of SIL: 11 low‐grade (LSIL) and 20 high‐grade (HSIL). Feulgen–pararosaniline staining was performed on previously Papanicolaou‐stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. Results: All 31 cases of SIL, either LSIL or HSIL, were non‐diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P = 0.32). Based on single‐cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P < 0.01). When a mean of 30% or more was used for the 6c‐exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single‐cell analysis than those with an indolent clinical behaviour: P = 0.04 and P = 0.03 for LSIL and HSIL, respectively. Conclusions: Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate ‘c’ values are used in single‐cell analysis. We propose that a >6c DNA content of 30% is useful as a cut‐off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.     

DNA ploidy of bladder cancer using bladder biopsy supernate specimens

OBJECTIVE: To perform DNA image cytometry on 119 bladder biopsy supernate (BBS) specimens of transitional cell carcinoma (TCC) bladder to: (1) test the suitability of this cytologic specimen for use in DNA ploidy analysis, and (2) assess the value of DNA ploidy measured on this specimen as to the risk of tumor recurrence and survival. STUDY DESIGN: The histologic grade and cytologic grade were correlated, and the DNA ploidy produced was determined by image analysis of Feulgen-stained nuclei. Kaplan-Meier curves related age, sex, grade and DNA ploidy to recurrence of tumor and survival. Log rank analyses were used to ascertain the difference between the curves for each categorical variable. RESULTS: Urothelial cells derived from the BBS specimen were demonstrated to be representative of the tumor. The tumor recurrence rate was significantly higher (P = .0001) and the survival rate significantly lower (P = .0002) for patients with aneuploid tumors compared to those with diploid tumors. Patients with TCC 2 tumors had a significantly shorter time to recurrence (P = .003), although the relationship between ploidy and survival in this group was of marginal significance. CONCLUSION: The specimen was free of many of the problems associate with the other specimen types used for measuring DNA ploidy. The results show that the BBS specimen is diagnostically useful and suitable for DNA analysis, providing prognostically relevant information.     

Roles of DNA cytometry and detection of EBERs in predicting a diagnosis of nasopharyngeal carcinoma.

OBJECTIVE: To analyze the suitability of DNA cytometry and detection of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) on nasopharyngeal brushings for predicting a diagnosis of nasopharyngeal carcinoma (NPC). STUDY DESIGN: Cytologic preparations in 66 cases suspicious for NPC were evaluated for NPC diagnosis in comparison with the histologic diagnosis. Based on cytologic examination, 38 cases containing cytologically proven cancer and 8 cases interpreted as cytologically negative for cancer with adequate cellularity in the smear specimens were analyzed for DNA ploidy with an image analyzer and for EBER expression by in situ hybridization (ISH). RESULTS: Based on the cytologic diagnosis, DNA aneuploidy analysis, DNA nondiploidy analysis and EBER detection demonstrated a sensitivity of 50%, 84% and 92%, respectively, with the same specificity, 100%, for predicting a diagnosis of cancer. Their negative predictive values were 30%, 57% and 73%, respectively. There was a significant difference between DNA aneuploidy analysis and EBER analysis in sensitivity (P < .001) and in negative predictive value (P < .05) but not between DNA nondiploidy analysis and EBER analysis even though EBER analysis showed a slightly higher value in both parameters (P > .1 and P > .5, respectively). CONCLUSION: ISH for EBERs in cytologic smears showed a role superior to that of DNA aneuploidy analysis in the diagnosis of NPC. Considering its advantages of simple experimental conditions and lower cost as compared with DNA measurement, EBER detection can play a practical and important diagnostic role in patients suspected of having primary NPC.     

DNA ploidy and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia in specimens obtained by punch biopsy

This study analyzed the feasibility of, and the strategy for, DNA ploidy analysis of cervical condyloma and intraepithelial neoplasia by a computerized digital imaging system. Paraffin-embedded tissue provided satisfactory single-cell preparations for DNA ploidy analysis after enzyme digestion and additional procedures. Negative endocervical curettings and normal squamous mucosa were used as internal diploid controls. With suitable controls, 21 (81%) of the 26 aneuploid lesions were identified as such in the single-cell preparations. The remaining five lesions (not recognized as aneuploid in the single-cell preparations) had ploidy levels between 2.08n and 2.30n and required DNA measurements on 12-microns sections. Criteria for these DNA measurements were defined: specimens intended for DNA ploidy analysis should contain abnormal epithelium of at least 3 mm to 4 mm in greatest dimension and should be accompanied by diploid controls, such as endocervical curettings or normal ectocervical squamous mucosa. With a combination of single-cell preparations and 12-microns tissue sections, it was possible to accurately determine the DNA ploidy patterns of the cervical lesion specimens obtained by punch biopsies. Available evidence suggests that ploidy analysis can provide useful diagnostic and prognostic information.     

High-risk HPV detection by Hybrid Capture and ploidy determination by a computer-assisted system in cervical biopsies

OBJECTIVE: To correlate high-risk HPV (hrHPV) detection by Hybrid Capture II (HC2) (Digene, Gaithersburg, Maryland, U.S.A.) with DNA content (ploidy) of cervical biopsies analyzed by a computer-assisted system. STUDY DESIGN: Cervical biopsies from 54 women examined at Leonor Mendes de Barros Hospital, S?o Paulo, as part of the Latin American Screening study during 2002--2003, were tested for hrHPV with HC2. All patients had been referred for colposcopic examination due to an abnormal cervical cytology. The final diagnosis included 30 cervicitis, 14 cervical intraepithelial neoplasia (CIN) 1, 5 CIN 2, 4 CIN 3 and 1 squamous cell carcinoma (SCC). Five-micrometer sections of each biopsy were stained with Feulgen-tionine and evaluated with the CAS 200 System (Becton Dickinson, U.S.A.), using the 3.0 software (version 8.1) of the DNA Quantitative Measurement Program (Becton Dickinson). Ploidy was evaluated from histograms obtained by analyzing atypical nuclei. RESULTS: Of the 30 cervicitis cases, 28 (93.3%) were diploid, and hrHPV was detected in 8 (28.5%) of the cases. Two tetraploid cervicitis lesions were observed, 1 positive and 1 negative for hrHPV. Among the CIN 1 lesions, 8 (57.1%) were diploid and 6 (42.8%) aneuploid. Of the latter, 4 (66.6%) were negative and 2 (33.3%) positive for hrHPV. Of the 5 CIN 2 lesions, 2 were diploid, 2 aneuploid and 1 tetraploid; all were positive for hrHPV. All CIN 3 lesions and the SCC proved to be aneuploid and positive for hrHPV. CONCLUSION: The data suggest that the majority of cervicitis and CIN 1 lesions are diploid and negativef or hrHPV. This is in sharp contrast to high grade CIN 2-3 lesions, all of which were positive for hrHPV in this study and also aneuploid, consistent with their progressive potential.